Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of phosphotyrosine, a central enzymatic reaction in eukaryotic signal transduction. Small molecule inhibitors that are specific for each cellular PTP would be valuable tools for dissecting phosphorylation networks and for validating PTPs as viable therapeutic targets. However, the common architecture of the conserved PTP protein fold impedes the design and discovery of selective PTP inhibitors. No general methods for specifically targeting a given PTP have been described. Because the human genome encodes more than 100 PTPs, the identification of inhibitors that are specific for each PTP through the standard methods of medicinal chemistry is not a practical prospect. This proposal describes the use of convergent engineering of enzyme/inhibitor interfaces to generate highly specific PTP inhibitors. The crux of this strategy resides in the design of "inhibitor-sensitized" PTPs-target PTPs that contain novel inhibitor-binding sites, which are not present in any wild-type PTP. The sensitizing pocket is introduced by mutating a functionally dispensable large amino acid residue to a smaller one (alanine or glycine). Specific inhibitors of the sensitized PTP are synthesized by modifying broad specificity inhibitors with bulky chemical groups. The appended substituents are designed to fit the novel active site pocket of the target enzyme and to diminish the potency of the inhibitors for wild-type PTPs. Successful complementary design, therefore, leads to binding interactions that are only possible in the engineered PTP/inhibitor complex. Transfection of cells with the gene encoding a sensitized PTP generates a biological system in which only one PTP can be blocked by the designed inhibitor. A key advantage of using mutagenesis to provide a unique molecular difference between the PTP of interest and all others is that the approach should be applicable across the superfamily. All human PTPs possess the conserved active site feature exploited by the proposed PTP/inhibitor interface engineering, as determined by primary sequence alignments. Therefore, this strategy could presumably be used to target any PTP in the genome.